Function Of Mobile Phase Composition Research Articles

Supercritical fluid chromatography with fluorescence detection for tocochromanol profiling in oils and seed ethanol extracts: A comparative study with NPLC

This study focused on the development of a method designed to simultaneously separate four tocopherol (T), four tocotrienol (T3) homologues and plastochromanol-8 (PC-8) using supercritical fluid chromatography (SFC) with fluorescence detection (FLD). We examined the selectivity and resolution behaviour of tocochromanols on eight different stationary phases and investigated the retention factor of PC-8 (kPC-8) as a function of mobile phase composition, as well as the injection volume’s impact on peak shape. The final SFC-FLD method developed involved a separation with two amino (NH2) columns connected in series (4.6 mm ID; 3 μm dp, 150 mm and 250 mm in length, respectively), at a flow rate of 2.5 mL/min, thermostated at 40 °C and a back-pressure regulator value of 10.0 MPa, injection volume of 10 μL and a gradient comprising of A:CO2 and B:MeOH. The sensitivity of SFC-DAD, SFC-FLD, NPLC-DAD and NPLC-FLD were compared and found to be 58.6–160.9, 5.9–51.9, 19.7–63.5 and 0.42–4.3 pmol, respectively. The SFC-FLD method developed was successfully applied to the analysis of seed ethanol extracts and plant oils and found to be suitable for the routine analysis. Furthermore, the SFC approach serves as a sustainable alternative compared to NPLC, due to the reduced human and environmental exposure and consumption of hazardous solvents.

Closed form approximations to predict retention times and peak widths in gradient elution under conditions of sample volume overload and sample solvent mismatch

Closed form expressions for the prediction of retention times and peak widths for gradient liquid chromatography are particularly useful in understanding, rationalizing and optimizing separations. These expressions are obtained by integrating differential equations, in conjunction with a model of the variation of the retention factor as a function of mobile phase composition. Two of these models, the linear solvent strength (LSS) model and the Neue-Kuss (NK) model are explored in the present work. Here, we expand on these closed form expressions to account for effects of sample volume overload and a mismatch between the sample solvent and the initial mobile phase composition for the gradient. We show that there have been errors in expressions reported in the literature, and we have evaluated the accuracy of the predictions from the closed form expressions reported here using a recently developed liquid chromatography simulator. The expressions assume a constant plate height and consider elution across four zones of the gradient profile – elution in the sample solvent, elution in the initial (isocratic) mobile phase caused by the gradient delay volume, elution during a linear gradient, and elution post-gradient at the final (isocratic) mobile phase composition. The expressions generally give reasonably accurate predictions for retention times and peak widths, except for cases where the solute elutes during transitions between the different zones. The average magnitude of the prediction errors for retention time and peak width relative to simulation were 0.093% and 0.40% for the LSS expressions for ten amphetamine solutes at 36 different separation conditions, and 0.22% and 1.8% for the NK expressions for eight alkylbenzene solutes at 36 different separation conditions, respectively.

Elucidation of retention behaviors in reversed-phase liquid chromatography as a function of mobile phase composition

Various retention models have been widely used for understanding the retention mechanisms of solutes in reversed-phase chromatography systems. The models have been used to interpret the often-observed linear plots of the logarithms of retention factor k versus the solvent modifier concentration CM and ln k versus ln⁡CM. In this study, the retention behaviors of nine solutes as a function of acetonitrile (ACN) concentration were systematically investigated using a commercially available C18 column. The thermodynamic properties of solute adsorptions in neat water, determined using van’ t Hoff plots, were investigated. Slightly concave upward and downward retention curves were identified for the plots of ln k versus CACN and ln k versus ln CACN, respectively. A three-equilibrium-constant stoichiometric displacement retention model was used to interpret the retention behaviors of the solutes. The model was demonstrated to account for the nonlinearity of the retention curves. The linear fits of the ln k versus ln⁡CACN and ln k versus CACN plots were implied to be more suitably used for high and low ACN concentration ranges, respectively. The model fitted the experimental data satisfactorily over a full range of ACN concentrations; thus, the nonlinearity of ln k versus ln⁡CACN plots was implied to mainly be attributed to the weak ACN–sorbent interactions. U-shaped retention curves were observed for acetone, tetrahydrofuran, tert-butanol, and benzyl alcohol at high ACN concentrations, indicating that the retention behaviors of these solutes may involve two types of interactions, with complementary effects on solute retention factor with increasing ACN concentration.

Evaluation of three temperature- and mobile phase-dependent retention models for reversed-phase liquid chromatographic retention and apparent retention enthalpy

Predicting retention and enthalpy allows for the simulation and optimization of advanced chromatographic techniques including gradient separations, temperature-assisted solute focusing, multidimensional liquid chromatography, and solvent focusing. In this paper we explore the fits of three expressions for retention as a function of mobile phase composition and temperature to retention data of 101 small molecules in reversed phase liquid chromatography. The three retention equations investigated are those by Neue and Kuss (NK) and two different equations by Pappa-Louisi et al., one based on a partition model (PL-P) and one based on an adsorption model (PL-A). More than 25 000 retention factors were determined for 101 small molecules under various mobile phase and temperature conditions. The pure experimental uncertainty is very small, approximately 0.22% uncertainty in retention factors measured on the same day (2.1% when performed on different days). Each of the three equations for ln(k) was fit to the experimental data based on a least-squares approach and the results were analyzed using lack-of-fit residuals. The PL-A model, while complex, gives the best overall fits. In addition to examining the equations’ adequacy for retention, we also examined their use for apparent retention enthalpy. This enthalpy can be predicted by taking the derivative of these expressions with respect to the inverse of absolute temperature. The numerical values of the fitted parameters based on retention data can then be used to predict retention enthalpy. These enthalpy predictions were compared to those obtained from a modified van ‘t Hoff equation that included a quadratic term in inverse temperature. Based on analysis of 1 211 van ‘t Hoff plots (solute-mobile phase-day combinations), ninety-eight percent showed a significantly better fit when using the modified van ‘t Hoff expression, justifying its use to provide apparent enthalpies as a function of mobile phase composition and temperature. The foregoing apparent enthalpies were compared to the apparent enthalpies predicted by the three models. The PL-A model, which contains a temperature dependent enthalpy, provided the best enthalpy prediction. However, there is virtually no correlation between the overall lack of fit to experimental ln(k) for each model and the corresponding lack of fit of the linear (in 1/T) van ‘t Hoff expression. Thus, the temperature-dependent enthalpy is apparently not the cause of a model’s ability to fit ln(k) as a function of mobile phase composition and temperature. The value in these expressions is their ability to predict chromatograms, allowing for optimization of an advanced chromatographic technique. The two simpler models NK and PL-P, which do not contain a temperature dependent enthalpy, have their merits in modelling retention (NK being the better of the two) and enthalpy (PL-P being the better of the two) if a simpler expression is required for a given application.

Modeling Retention Behavior on Analysis of Hallucinogenic Mushrooms Using Hydrophilic Interaction Liquid Chromatography.

The goal of this work was to investigate and compare the selectivity of three different hydrophilic interaction liquid chromatography (HILIC) charge modulated amide columns, iHILIC®-Fusion, iHILIC®-Fusion(+) and iHILIC®-Fusion(P), for analysis of compounds in hallucinogen mushrooms. An extract of a truffle-like fungus containing psilocin, psilocybin and baeocystin was chosen as test material. Three different modeling methods were applied to describe the retention times of constituents in isocratic separation mode as a function of mobile phase composition, pH and temperature. Two models using DryLab® 2010 assumed quadratic and exponential relationship between the retention time and solvent fraction of aqueous component of the mobile phase, respectively. These models also illustrate the van't Hoff like equation to describe the temperature-dependence of the retention factor and the theory of Snyder et al. to estimate the retention factor as a function of pH of the aqueous mobile phase component. The third model using STATISTICA® multivariate data analysis in a predefined experimental space was able to predict the retention times. All HILIC columns in this comparison were proved to be suitable for separation of the two hallucinogenic alkaloids from each other and from the matrix components. Majority of compounds were separated with satisfactory resolutions required by the comparative analysis despite some of them were not fully baseline separated. It was found the best modeling was obtained by using the quadratic approach to predict chromatograms for predefined chromatographic conditions (volumetric ratio of acetonitrile to buffer, pH of the buffer and temperature), while the exponential model proved to be the worst for prediction. The modeling with multivariate data analysis fell between the other two methods.

Validation and Peak Purity Assessment of RP-HPLC Method for Simultaneous Estimation of Amitriptyline HCl, Chlordiazepoxide and Comparison with Spectrophotometry

Purpose : The study was performed with the aim to design a Simple, precise and economical RP-HPLC method have been developed for simultaneous estimation of amitriptyline HCl and chlordiazepoxide in pharmaceutical formulation. Approach : RP-HPLC was developed using WATERS C18 250 mm × 4.6 mm (5 μm) column, utilizing a mobile phase of methanol: acetonitrile: 0.2 M orthophosphoric acid, pH 4.5 (adjusted with triethylamine) 56:24:20 ml (with a flow rate of 1ml/min) and U.V detection was carried out at 240nm. Optimization of this method was carried out as a function of mobile phase composition, pH and flow rate. Peak purity was assessed by spectral comparison method. Findings : RP-HPLC method shows linearity in the range of for 0.05-1.6 μg/ml and 0.01-0.32μg/ml amitriptyline HCl and chlordiazepoxide respectively. The LOD and LOQ values for amitriptyline HCl were 0.0250μg/ml and 0.0759 μg/ml and 0.0045 μg/ml and 0.0135 μg/ml for chlordiazepoxide. The results of % purity was found to be 99.97 ± 0.58 % w/w and 99.29 ± 0.59 % w/w for HPLC method and 98.87 ± 0.03 % w/w and 98.81 ± 1.04 % w/w for first derivative spectrophotometric method for amitriptyline HCl and chlordiazepoxide respectively. Conclusion : Results of descriptive statistics studies conclude that RP-HPLC method found to be more sensitive and convenient than first derivative spectrophotometric method.

Revisiting molecular weight distribution of polystyrenes using adsorption high-performance thin-layer chromatography

A repeatable separation of polystyrenes according to MW, from 1920 to 520000 u.m.a, has been obtained under adsorption conditions using a method that comprises the use of Lichrospher HPTLC plates, and a controlled, isocratic elution with a 78:22 (v/v) mixture of cyclohexane (Cy)-tetrahydrofuran (THF). Likewise, UV-densitometric quantification of polystyrenes in mixtures can be achieved, by an intra-plate or an inter-plate procedure, using the corresponding polystyrene calibration curve. In the case or overlapped, unresolved peaks, an average curve of the corresponding polystyrenes can be used. Migration of polystyrenes strongly varies with slight variations in the relative proportion of Cy and THF in the mobile phase. This allows different ranges of MW to be separated as a function of mobile phase composition. Other factors influencing repeatability have been identified. Some reasons have been advanced to explain the current lack of activity in the research on polymer characterization by HPTLC. Old literature results concerning polystyrene separation have also been discussed in the light of modern HPTLC instrumentation. Developed method provides similar information on Molecular Weight Distribution (MWD) to that obtained using Gel Permeation Chromatography (GPC). Advantages and limitations of HPTLC for obtaining polymer MWD have also been discussed.

Correction of the deviations in the retention times with Chromolith columns associated to the flow rate: Implications in the modelling of the retention behaviour

In a previous work (J. Sep. Sci. 2009, 32, 2793-2803), we reported an interpretive optimisation approach to achieve maximal resolution in minimal analysis time, based on models describing the retention and peak shape as a function of mobile phase composition and flow rate. The method was applied to the separation of a group of basic drugs in a Chromolith column. In that work, we found that the retention factors were sensitive to the flow rate. The reason of the observed deviations in retention times is the increase in the column volume at the applied pressure, which decreases the linear velocity inside the column. This behaviour forced to include a correction term in the model that described the retention. We show here how the deviations in retention times can be evaluated, allowing retention models that do not include the flow rate as a variable, similar to isocratic chromatography at fixed flow rate. The logarithm of the deviations in the retention times with flow rate is shown to correlate with the solute polarity. This correlation is compared with similar correlations for the retention factor at fixed mobile phase composition and the extrapolated retention factor in water at fixed flow rate.

Book Review: Ion-Pair Chromatography and Related Techniques

Book Review: Ion-Pair Chromatography and Related Techniques

Quantitative structure–retention relationships of pesticides in reversed-phase high-performance liquid chromatography based on WHIM and GETAWAY molecular descriptors

The ability of the Weighted Holistic Invariant Molecular (WHIM) and GEometry, Topology, and Atom-Weights AssemblY (GETAWAY) descriptors to represent the effect of molecular structure on the retention of pesticides in reversed-phase high-performance liquid chromatography (RP-HPLC) is investigated. To this end, two retention data sets previously collected in water–acetonitrile mobile phase are re-examined. The first data set ( data-set-1) consists of retention data of 26 neutral compounds belonging to widely used pesticide classes, collected within the mobile phase composition range 40–65% (v/v) acetonitrile. The second data set ( data-set-2) describes retention of phenoxy acid herbicides and structurally related compounds (benzoic acid and phenylacetic acid derivatives), as a whole covering the p K a range 2.3–4.3, as a function of mobile phase composition, ranging between 30 and 70% (v/v) acetonitrile, and pH, ranging between 2 and 5. For each data set, the mobile phase attributes are combined with WHIM or GETAWAY descriptors into “mixed” predictive models in order to attempt retention modelling within the whole mobile phase composition range of analytical interest. Six- or seven-dimensional multilinear models, preliminarily selected using a genetic algorithm, were improved using a multi-layer artificial neural network (ANN) learned by back propagation. ANN performance was tested on three molecules not used in the learning stage and by leave-more-out cross validation. The results reveal that while WHIM descriptors seem not adequate to model retention of solutes of data-set-1, GETAWAY descriptors provide a satisfactory retention model. On the other hand WHIM and GETAWAY descriptors applied to data-set-2 provide a similar performance, even if slightly worse as compared with the above case. Accuracy of retention modelling in these cases is comparable or slightly poorer as compared with the results previously obtained by combining quantum chemical descriptors or usual physico-chemical solute properties (log k ow and p K a) and mobile phase attributes.

Comparative study of solvation parameter models accounting the effects of mobile phase composition in reversed-phase liquid chromatography

Solvation parameter models relate linearly compound properties with five fundamental solute descriptors (excess molar refraction, dipolarity/polarizability, effective hydrogen-bond acidity and basicity, and McGowan volume). These models are widely used, due to the availability of protocols to obtain the descriptors, good performance, and general applicability. Several approaches to predict retention in reversed-phase liquid chromatography (RPLC) as a function of these descriptors and mobile phase composition are compared, assaying the performance with a set of 146 organic compounds of diverse nature, eluted with acetonitrile and methanol. The approaches are classified in two groups: those that only allow predictions of retention for the mobile phases used to build the models, and those valid at any other mobile phase composition. The first group includes the use of ratios between the regressed coefficients of the solvation models that are assumed to be characteristic for a column/solvent system, and the application of offsets to transfer the retention from a reference mobile phase to any other. Maximal accuracy in predictions corresponded, however, to the approaches in the second group, which were based on models that describe the retention as a function of mobile phase composition (expressed as the solvent volume fraction or a normalised polarity measurement), where the coefficients were made dependent on the solvent descriptors. The study revealed the properties that influence the retention and distinguish the particular behaviour of acetonitrile and methanol in RPLC.

Prediction of peak shape in hydro-organic and micellar-organic liquid chromatography as a function of mobile phase composition

A simple model is proposed that relates the parameters describing the peak width with the retention time, which can be easily predicted as a function of mobile phase composition. This allows the further prediction of peak shape with global errors below 5%, using a modified Gaussian model with a parabolic variance. The model is useful in the optimisation of chromatographic resolution to assess an eventual overlapping of close peaks. The dependence of peak shape with mobile phase composition was studied for mobile phases containing acetonitrile in the presence and absence of micellised surfactant (micellar-organic and hydro-organic reversed-phase liquid chromatography, RPLC). In micellar RPLC, both modifiers (surfactant and acetonitrile) were observed to decrease or improve the efficiencies in the same percentage, at least in the studied concentration ranges. The study also revealed that the problem of achieving smaller efficiencies in this chromatographic mode, compared to hydro-organic RPLC, is not only related to the presence of surfactant covering the stationary phase, but also to the smaller concentration of organic solvent in the mobile phase.

Predicting the chromatographic retention of polymers: Poly(methyl methacrylate)s and polyacryate blends

The suitability of a retention model especially designed for polymers is investigated to describe and predict the chromatographic retention behavior of poly(methyl methacrylate)s as a function of mobile phase composition and gradient steepness. It is found that three simple yet rationally chosen chromatographic experiments suffice to extract the analyte specific model parameters necessary to calculate the retention volumes. This allows predicting accurate retention volumes based on a minimum number of initial experiments. Therefore, methods for polymer separations can be developed in relatively short time. The suitability of the virtual chromatography approach to predict the separation of polymer blend is demonstrated for the first time using a blend of different polyacrylates.

Optimization of artificial neural network for retention modeling in high-performance liquid chromatography

An artificial neural network (ANN) model for the prediction of retention times in high-performance liquid chromatography (HPLC) was developed and optimized. A three-layer feed-forward ANN has been used to model retention behavior of nine phenols as a function of mobile phase composition (methanol-acetic acid mobile phase). The number of hidden layer nodes, number of iteration steps and the number of experimental data points used for training set were optimized. By using a relatively small amount of experimental data (25 experimental data points in the training set), a very accurate prediction of the retention (percentage normalized differences between the predicted and the experimental data less than 0.6%) was obtained. It was shown that the prediction ability of ANN model linearly decreased with the reduction of number of experiments for the training data set. The results obtained demonstrate that ANN offers a straightforward way for retention modeling in isocratic HPLC separation of a complex mixture of compounds widely different in pKa and logKow values.

Retention characteristics of protonated mobile phases injected into deuterated mobile phases in capillary liquid chromatography (LC) using on-line nuclear magnetic resonance (NMR) detection.

The behavior of protonated binary solvents injected into deuterated binary mobile phases in capillary LC is studied with NMR. Specifically, the solvent elution is followed on-flow with a capillary LC coupled to a 900 nL volume microcoil NMR probe. A range of identical composition 5% protonated (and 95% deuterated) solvents is injected into composition-matched deuterated mobile phases of CD(3)CN/D(2)O and CD(3)OD/D(2)O. The protonated components separate for all solvent combinations except at 80% CD(3)CN/20% D(2)O and similar to 72% CD(3)OD/28% D(2)O where only a single retention time is observed. The more hydrophilic protonated component, HOD, elutes first with higher percentages of hydrophilic solvent, D(2)O, in the mobile phase whereas retention is reversed with the higher percentage of the more hydrophobic solvent (CD(3)CN and CD(3)OD) in the mobile phase. The hydrophilic/hydrophobic nature of the chromatographic system as a function of mobile phase composition is characterized by following the retention times of protonated solvents.

Prediction of peak shape as a function of retention in reversed-phase liquid chromatography

Optimisation of the resolution of multicomponent samples in HPLC is usually carried out by changing the elution conditions and considering the variation in retention of the analytes, to which a standard peak shape is assigned. However, the change in peak shape with the composition of the mobile phase can ruin the optimisation process, yielding unexpected overlaps in the experimental chromatograms for the predicted optimum, especially for complex mixtures. The possibility of modelling peak shape, in addition to peak position, is therefore attractive. A simple modified-Gaussian model with a parabolic variance, which is a function of conventional experimental parameters: retention time ( t R), peak height ( H 0), standard deviation at the peak maximum ( σ 0), and left ( A) and right ( B) halfwidths, is proposed. The model is a simplification of a previous equation proposed in our laboratory. Linear and parabolic relationships were found between the peak shape parameters ( σ 0, A and B) and t R, with a mean relative error of 1–5% in most cases. This error was partially due to variations in peak position and shape among injections, which in some cases were above 2%. Correlations between ( σ 0, A and B) and the retention time, which is easily modelled as a function of mobile phase composition, allowed a simple and reliable prediction of chromatographic peaks. A parameter that depends on the slopes of the linear relationships for A and B versus t R is also proposed to evaluate column efficiency. The modified-Gaussian model was used to describe the peaks of six diuretics of diverse acid–base behaviour and polarity, which were eluted with 15 mobile phases where the composition was varied between 30 and 50% (v/v) acetonitrile and the pH between 3 and 7.

Evaluation of a reversed-phase column (supelcosil LC-ABZ) under isocratic and gradient elution conditions for estimating octanol-water partition coefficients.

The solvation parameter model is used to identify suitable chromatographic models for estimating the octanol-water partition coefficient for neutral compounds of varied structure by reversed-phase liquid chromatography. The stationary phase Supelcosil LC-ABZ with methanol-water mobile phases affords a series of suitable correlation models for estimating the octanol-water partition coefficient (log KOW) under isocratic and gradient elution conditions. Isocratic separations with mobile phase compositions containing from about 25 to 40% (v/v) methanol provide the most accurate results for log KOW values in the range -0.1 to 4.0. Gradient separations programmed from 5 to 100% (v/v) methanol are suitable for faster separations of compounds with large log KOW values. The standard error in the estimate for the regression models of the predicted log KOW values against literature values are 0.135 log units for the 30% (v/v) methanol-water isocratic system and 0.263 log units for the methanol-water gradient system. Isocratic retention factors predicted from two gradient separations with gradient times of 15 and 45 min afford a poorer fit for the correlation models between log KOW and the estimated retention factors than that of either the above isocratic and gradient models. Plots of the retention factor (log k) as a function of mobile phase composition are generally non-linear. Values of log kw obtained by non-linear extrapolation to a volume fraction of 0% (v/v) methanol do not afford a useful model for estimating log KOW.

Chromatographic shape selectivity with carbon dioxide–acetonitrile mobile phases: Effect of mobile phase composition and density

Trends in chromatographic shape selectivity with mobile phases consisting of mixtures of carbon dioxide and acetonitrile are investigated. Selectivity is evaluated as a function of mobile phase composition, temperature, and column bonding chemistry. SRM (standard reference material) 869a is used as a probe of shape selectivity, while the selectivity between triphenylene and o-terphenyl is used to investigate planarity selectivity. Four molecular mass 228 polyaromatic hydrocarbon isomers are used to investigate shape selectivity based on differences in length-to-breadth ratio. Shape selectivity trends as a function of temperature and column type are found to be similar to what is seen in reversed-phase liquid chromatography, while the trend seen as the amount of acetonitrile in the mobile phase increases is found to be different than in reversed-phase liquid chromatography. In addition, the effect of mobile phase density, i.e., solvent strength, on shape selectivity is investigated by examining shape selectivity as a function of density with neat carbon dioxide as the mobile phase.

A study of the mechanisms involved in the separation of metal ions with a mixed-bed stationary phase

The optimization of chromatographic methods for the determination of metal species require an understanding of the mechanisms involved. In this work, the separation of Cd, Co, Cu, Fe(II/III), Mn, Pb and Zn using a mixed bed column (IonPac CS5A) and a cation-exchange column (IonPac CS2) is studied as a function of mobile phase composition. The type and concentration of complexing agent and of ionic strength modificators were evaluated. The charge of analytes were calculated using the classical ion exchange approach to highlight the effect of eluent composition on retention. The comparative study enabled us to identify an optimal eluent composition for the separation of the nine metal species.

Solid-state NMR characterization of amylose tris(3,5-dimethylphenylcarbamate) chiral stationary-phase structure as a function of mobile-phase composition.

Solid-state NMR (1H/13C CPMAS) was utilized to identify structural differences in amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase (Chiralpak AD), as a function of mobile-phase composition. Dry Chiralpak AD stationary phase displayed an amorphous CPMAS NMR spectrum. However, CPMAS spectra of Chiralpak AD flushed with organic mobile phases clearly displayed evidence of solvent complexes. Chiralpak AD flushed with nonpolar hexane exhibited solvent complexes with minimal structural perturbation. For Chiralpak AD flushed with hexane containing alcohol modifiers, however, solvent incorporation caused significant difference in conformation distribution as evidenced by increased resolution of 13C peaks in the CPMAS spectrum of the stationary phase. 2-Propanol modifier displayed more efficient displacement of incorporated hexane while forming relatively more distinct/ordered solvent complexes with Chiralpak AD in comparison to ethanol modifier. Reversed elution order and unusual retention behavior on Chiralpak AD as a function of mobile-phase modifier was reported earlier. These chromatographic behaviors are believed to be due to different alterations of the steric environment of the chiral cavities in the CSP by the different mobile-phase modifiers. In addition, on the basis of the chemical shift of C-1 carbon on the amylose backbone, it is possible that Chiralpak AD's structure is a helix with a number of fold less than six.